Potassium channels are specialized integral membrane proteins endowed with a remarkable capacity to accommodate the highly selective passage of potassium ions across cells. Some have the added ability to open and close in response to small changes in transmembrane voltage, and it is these voltage-gated (Kv) potassium channels that are the focus of this proposal. These proteins play crucial roles in a number of physiologic processes ranging from the propagation of information in the nervous system to the maintenance of a normal heart rhythm, and inherited mutations in many of them lead to forms of epilepsy, paralysis, and cardiac arrhythmias. The structures of some parts of Kv channels and their auxiliary subunits are now well understood, yet despite detailed study for over a decade, little is known about the construction of the voltage sensing region of these channels, or about the overall architecture of hetermultimeric channels. In light of this gap, a new method is introduced to complement established techniques of molecular biology and electrophysiology--the use of tethered quaternary ammonium blockers as molecular tape-measures. These compounds will be targeted to intracellular and extracellular regions of two classes of K+ channels: the prototypic voltage-dependent Shaker channel, and heteromultimeric channels formed from the co-assembly of Shaker-like subunits with minK-related peptides (MiRPs). The two specific aims, (1) mapping the extracellular portion of Shaker's gating module, and (2) probing the structure of MiRP-associated channels, will be instrumental in fulfilling the project's long-term objectives of creating a detailed physical map of the gating module of a Kv channel, and determining how the different parts of a Kv channel are molded together.